Novel components of iron sulfur cluster biosynthesis pathways

描述（由申请人提供）：铁硫（FeS）簇是古老的普遍存在的金属蛋白辅因子，其起源被认为是在早期厌氧生物圈的还原环境中。向有氧生活的过渡已经为FeS簇产生了几个问题，因为氧化限制了铁的生物利用度，并且直接促进了簇与氧物种如超氧化物和过氧化物的破坏，这是有氧呼吸的副产物。这种易感性已被宿主防御机制利用，其靶向FeS簇作为促进细胞死亡的组分。FeS簇的组分铁和硫对细胞具有高度毒性，并且FeS簇通过特定的生物合成途径形成，例如大肠杆菌中的iscRSUhscBAfdx操纵子和sufABCDSE操纵子。我们的长期目标是确定一套完整的因素参与FeS簇生物合成的模式原核生物E。大肠杆菌和功能特征的FeS簇的形成中的作用。这项研究的目的是鉴定一种新的因子，CGFS型单硫醇谷氧还蛋白，GrxD，它与E。coli Suf FeS生物合成途径，并测定其在E.杆菌Suf途径似乎已经适应了在胁迫条件下（如氧化胁迫和铁限制）在FeS簇合成中的作用，并且已经被证明是植物病原体欧文氏菌（Erwinia chalcii）的完全毒力所必需的。此外，FeS簇生物合成过程是一个重要特征，Suf系统已被确定为某些细菌物种中唯一的FeS簇生物合成途径，例如结核病病原体结核分枝杆菌（结核病的病原体）。结核病每年造成大约200万人死亡，主要是在发展中国家。Suf系统的特点是在一些细节，在最近几年，然而，研究的Isc和Suf系统还没有确定具体的适应，使Suf能够在应力条件下的FeS簇合成。此外，最近鉴定的因素以外的这两个核心操纵子，参与FeS簇的生物合成，表明其他因素不编码的Suf操纵子，如GrxD，可能与Suf系统和促进合成的FeS簇在应力条件下。使用生物化学测定、功能基因组学、蛋白质化学和分子生物学的组合，我们的目标是（i）确定grxD和Isc系统突变体之间的合成致死性的基础，以及这些遗传相互作用是否可以在特定条件下减轻，（ii）表征GrxD的物理相互作用并确定其参与的蛋白质复合物的组成，和GrxD的生化特性的结合伙伴的影响，（iii）具体评估GrxD转移其FeS簇的能力，和（iv）确定的功能关联的GrxD的Suf和Isc系统。

DESCRIPTION (provided by applicant): Iron-sulfur (FeS) clusters are ancient ubiquitous metalloprotein cofactors whose origins are thought to lay in the reducing environment of the early anaerobic biosphere. The transition to aerobic life has created several problems for FeS clusters by both limiting the bio-availability of iron due to oxidation and directly promoting the destruction of clusters with oxygen species such as superoxide and peroxide, which are by-products of aerobic respiration. This susceptibility has been leveraged by host defense mechanisms, which target FeS clusters as components through which to promote cell death. The components of FeS clusters, iron and sulfur, are highly toxic to cells and FeS clusters are formed by specific biosynthesis pathways such as the iscRSUhscBAfdx operon and sufABCDSE operon in Escherichia coli. Our long term goal is to determine a complete set of factors involved in FeS cluster biosynthesis in the model prokaryote E. coli and to functionally characterize their roles in FeS cluster formation. The objective of the proposed research is the characterization of a novel factor, the CGFS-type monothiol glutaredoxin, GrxD, which has been implicated as functioning with the E. coli Suf FeS biosynthesis pathway, and the determination of its role in E. coli. The Suf pathway appears to have adapted to a role in FeS cluster synthesis under stress conditions, such as oxidative stress and iron limitation, and has been shown to be necessary for complete virulence of the plant pathogen Erwinia chrysanthemi. Moreover, the process of FeS cluster biosynthesis is an essential trait, and the Suf system has been identified as the only FeS cluster biosynthesis pathway in some bacterial species, such as the pathogen Mycobacterium tuberculosis, the causative agent of tuberculosis. Tuberculosis kills approximately 2 million people a year, mainly in the developing world. The Suf system has been characterized in some detail in recent years, however, studies of both Isc and Suf systems have yet to identify specific adaptations which make Suf capable of FeS cluster synthesis under stress conditions. Moreover, recent identification of factors outside of these two core operons, which participate in FeS cluster biosynthesis, suggests that other factors not encoded in the Suf operon, such as GrxD, may work with the Suf system and facilitate the synthesis of FeS clusters under stress conditions. Using a combination of biochemical assays, functional genomics, protein chemistry and molecular biology our aims are to (i) determine the basis of synthetic lethality between grxD and Isc system mutants, and if these genetic interactions can be alleviated under specific conditions, (ii) characterize the physical interactions of GrxD and determine the composition of protein complexes in which it participates, and the effect of binding partners on the biochemical properties of GrxD, (iii) specifically assess the ability of GrxD to transfer its FeS cluster, and (iv) determine the functional association of GrxD to both Suf and Isc systems.